Alphaviruses are transmitted by mosquitos and cause encephalitis in animals and man. A prototype alphavirus, Sindbis virus, infects and kills most cultured cell types, not by lethal parasitism, but by inducing programmed cell death with apoptotic morphology. Similarly, Sindbis virus induces apoptosis in central nervous system neurons of newborn mice which are killed by the virus, but has a reduced ability to induce apoptosis in mosquito cells and in neurons of older mice which survive infection. Thus, the ability of Sindbis virus to induce apoptosis of neurons is a significant factor in viral pathogenesis as the induction of neuronal apoptosis correlates with mortality. These and other findings raise the interesting possibility that host cell factors that regulate the programmed cell death pathway can modulate the outcome of a Sindbis virus infection. To explore the role of cellular death/survival genes involved in Sindbis virus-induced apoptosis, a Sindbis virus vector system was developed. The Sindbis virus vector serves both as a neuron- specific targeting vehicle to express foreign genes, and as a viral trigger to activate the cellular death pathway. In addition, the Sindbis virus vector system allows quantitative assessment of the ability of foreign genes to modulate viral pathogenesis in a mouse model. Expression of Bcl-2, a cellular apoptosis inhibitor, or CrmA, a viral inhibitor of cellular caspases, via the Sindbis virus vector protects newborn mice from a fatal Sindbis virus infection, supporting the hypothesis that apoptosis regulators can alter the course of a virus infection. In this study, the Sindbis virus vector system and genetically deficient mice will be used to study the molecular mechanisms by which Bcl-2 family members and caspases modulate viral pathogenesis. Preliminary results show that Bcl-2 family members have altered functions in neurons and these functions will be further delineated by targeted mutagenesis of Bcl-2 family proteins, mouse mortality studies, and measurements of neuronal apoptosis and viral replication efficiencies. The downstream effectors of these molecular pathways will be sought. Delineation of the caspase cascade in Sindbis virus-induced neuronal apoptosis will be evaluated by expression of dominant negative caspases via the Sindbis virus vector and through other biochemical approaches. These studies are expected to delineate the molecular mechanisms by which cellular factors modulate Sindbis virus neurovirulence, and these findings are likely to apply to other viral systems. In addition, an increased understanding of the neuronal cell death pathway has ramifications for normal cellular processes as well as neurodegenerative disease.